The present invention relates to a method for transcription of a DNA sequence and, more particularly, to a method for transcription of the DNA sequence suitable for obtaining information of crude data detected as it is by a DNA sequencing apparatus using the fluorescent method. The present invention further relates to an apparatus for transcription of the DNA sequence adapted to acquire and record crude data of the DNA sequence detected by the DNA sequencing apparatus as it is.
The DNA sequencing apparatus using the fluorescent method has the advantage that a dangerous and expensive radioactive isotope is not required any more.
DNA sequencing on the basis of the fluorescent method is generally executed by electrophoresis using DNA fragments which are labelled with a fluorescent substance. The DNA fragments may be prepared by breaking down a DNA sequence sought to be subjected to sequence determination with restriction reagents in such a manner that bands of bases of various lengths are provided at terminals each with a specific one of four bases selected from a group of adenine (A), cytosine (C), guanine (G) and thymine (T) while controlling reactivities of chemical reactions specific to sites of base linkages. These fragments may be isolated by electrophoresis in accordance with lengths of bands of bases so that sequences of bases in the bands of bases can be determined from a distribution of intensities of fluorescence generated from the fluorescent substance labelled on each of the DNA fragments upon excitation.
An example of a distribution of DNA fragments after electrophoresis is shown in FIG. 9. As distances of electrophoresis vary with lengths of DNA fragments, it is to be noted that DNA fragments of identical molecular weights are gathered as time lapses, thus forming bands 91 corresponding to molecular weights and giving a distribution pattern of DNA as a whole as shown in FIG. 9. The bands in lanes 92, 93, 94 and 95 of respective bases, A, C, G, and T, are not located horizontally in a row because distances of electrophoresis vary from the other lanes due to a difference in molecular weights from the next band by more than one base.
A distribution pattern of a DNA fragments labelled with fluorescent substances may be produced by exposing a gel to laser beams or the like and detecting fluorescence generated upon reaction by a photoelectric converting element, while a distribution pattern of DNA fragments labelled with a radioactive substance may be irradiated on an X-ray film.
An electrophoresis apparatus adapted to detect a DNA sequence using the fluorescent detection method of the above type is described, for example, in Japanese Patent Publication (laid-open) No. 62,843/1986.
An electrophoresis apparatus is described in conjunction with FIG. 7 in which an electrophoresis apparatus 70 using the fluorescent method comprises an optical source 71 for exciting fluorescence, an optical fiber 72 for leading a light from the optical source 71, an electrophoresis unit 73 for carrying out electrophoresis, an upper electrode 74a and a lower electrode 74b for applying voltages to the electrophoresis unit 73, an electric source unit 75 for applying voltages to the electrodes, a lens system 76 for receiving fluorescence, an optical amplifier 77 for amplifying an optical signal, a one-dimensional optical sensor 78 for converting an image optically amplified into an electric signal, and a signal line 79 for supplying the electric signal from the optical sensor 78. Electric signals of a distribution pattern of a DNA fragment detected by the electrophoresis apparatus 70 are fed from the signal line 79 to a sequencing apparatus 80 in which the distribution pattern is sequenced and the resultant data is given. The sequencing apparatus 80 comprises an amplifier 81 for amplifying the electric signal fed from the signal line 79 in response to the electric signals of the distribution pattern of the DNA fragment detected by the electrophoresis apparatus 70, an analog-to-digital converting circuit 82 for converting an electric signal from analog to digital signals, a processing unit 83 for determining a sequence of bases from a digitized signal line, and an output unit 84 for outputting a signal line of the resultant base sequence.
Operation of the electrophoresis apparatus having the above structure will be described in conjunction with FIGS. 7, 8A, and 8B.
FIGS. 8A and 8B are a front view and a longitudinally cross-sectional view of the electrophoresis unit 73, respectively, which is shown to be constituted by a pair of glass plates 73b arranged so as to interpose a gel 73a, such as polyacrylamide, between both plates. A sample of DNA fragments is fed from an upper portion of the gel 73a of the electrophoresis unit 73 and the electrophoresis is executed by applying voltages from the upper and lower electrodes. A light, for example a laser beam, from the optical source 71 is radiated at an optical path 85 from the lower left-hand side in the drawing to the gel 73a of the electrophoresis unit 73 through the optical fiber 72. A fluorescent body of the DNA fragment in the gel 73a on the optical path 75 is excited to generate fluorescence 86.
The fluorescence 86 generated from the optical path 85 in the electrophoresis unit 73 is gathered and led to the optical amplifier 77 where an intensity of the light is amplified several thousand times to several tens of thousands of times. The amplified light is led to the one-dimensional optical sensor 78 where it is converted into electric signals. The one-dimensional optical sensor 78 is an optical sensor which is arranged in one-dimension so as to provide an image along the optical path 85. Electric signals as analog signals provided by the one-dimensional optical sensor 78 are then amplified by the amplifier 81 and digitized in the analog-to-digital (A/D) converting circuit 82. The digitized signals are fed to the processing unit 83 where a variation in fluorescent intensities at a virtually middle position of each of the lanes 92, 93, 94 and 95 on the optical path 85 is measured so as to judge whether a band is present or not, thus determining a sequence of bases consisting of A, C, G, and T. A sequence of the bases determined is converted into symbols such as letter codes or the like and printed out by a printer 84.
As has been described hereinabove, the DNA sequencing on the basis of the fluorescent method involves labelling DNA fragments with fluorescent substances, transferring a gel containing the labelled DNA fragments between a light emitting element of a laser and a photoelectric transfer element after electrophoresis, detecting fluorescence generated upon reaction with the laser beam or the like from the gel by the optoelectric transfer element, processing the detected data by means of the analog-to-digital conversion or other techniques to execute digital processing, and outputting only particular data corresponding to each base as a DNA sequence data with the printer or the like as letter data.
It is noted, however, that a conventional DNA sequencing apparatus for executing the DNA sequencing on the basis of the fluorescent method pays no great attention to the fact that a crude data of DNA sequences appearing on the gel as a fluorescent image can be left as an exhibit for evidence. It is further designed so as to digitize data detected by a photoelectric transfer element and output only particular data so that it suffers from the disadvantages that confirmation is impossible even if there is an error the processed results, the results may be falsified readily, and the competency of the results as scientific evidence is low. Further, prior art DNA sequencing apparatuses are made larger due to digital conversion of the data, digital analysis and the like and thus are rendered expensive.